Abstract

A1 adenosine receptors inhibit adenylate cyclase by activating Gi/Go, whereas A2A receptors activate Gs. We examined how regions of A1 and A2A receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to Gi/Go and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A1/A2A 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A1 3ICL sequence (A2A/A1L or A2A/A1LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to Gi/Go. These chimaeras have basal cAMP levels lower than those of wild-type A2A receptors, similar to wild-type A1 receptors. The A1 C-terminus modulates the coupling of receptors with A1 3ICL such that A2A/A1LT is better coupled to Gi/Go than A2A/A1L. The C-terminus has little impact on coupling to receptors containing A2A 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to Gi/Go mediated by A1 3ICL and not by other intracellular domains that favour Gi coupling. The C-terminus sequence has little or no effect on coupling to Gs. For doubly Gs/Gi-coupled adenosine receptors in HEK-293 cells, Gs-mediated stimulation predominates over Gi/Go-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.